Portions of the eukaryotic genome can be maintained in a transcriptionally inactive, or silenced, state as the result of the local chromatin structure. Silent chromatin may encompass regions ranging from a few thousand base pairs, as in the silent mating type genes of the yeast S. cerevisiae (Loo, S. & Rine, J. (1995) Annu. Rev. Cell Dev. Biol. 11, 519-48), to whole chromosomes, such as the inactive X-chromosome in mammals (Lyon, M. F. (1999) Curr. Biol. 9, R235-7). The formation of silent chromatin, which is best understood at the S. cerevisiae silent mating type loci HMR and HML, and telomeres, depends on DNA elements, or silencers. The HM silencers are located in proximity to the genes they regulate and contain a combination of binding sites for Raplp, Abflp and the origin recognition complex (ORC) (Loo, S. & Rine, J. (1995) Annu. Rev. Cell Dev. Biol. 11, 519-48). These proteins recruit the SIR (Silent Information Regulator) protein complex (Sir2p-4p) through protein-protein interactions. Once recruited to silencers, the SIR complex is thought to spread along the chromatin through binding of Sir3p and Sir4p to the NH2-terminal tails of histone H3 and H4 (reviewed in Gartenberg, M. R. (2000) Curr. Opin. Microbiol. 3, 132-7). Among the many requirements for silent chromatin (reviewed in Wu, J. & Grunstein, M. (2000) Trends Biochem. Sci. 25, 619-23), post-translational modification (i.e. acetylation, phosphorylation, methylation and ubiquitination) of the NH2-terminal tails of histones appears to be critical. For example, the tails of histones H3 and H4 are hypoacetylated in silent chromatin compared to other regions of the genome (Braunstein, M. et al. (1993) Genes Dev. 7, 592-604). Of the SIR proteins, Sir2p has been shown to be an NAD+-dependent histone deacetylase, and is responsible for the hypoacetylated state of histones in silent chromatin (Moazed, D. (2001) Curr. Opin. Cell Biol. 13, 232-8; Imai, S. et al. (2000) Nature 403, 795-800; Smith, J. S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 6658-63; Landry, J. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 5807-11). Sir2p also acts at the ribosomal RNA gene cluster (rDNA) in the RENT protein complex, which does not include Sir3p or Sir4p (Straight, A. F. et al. (1999) Cell 97, 245-56), where it acts to repress recombination.
The yeast SIR2 gene is the defining member of a broadly conserved family of NAD+-dependent deacetylases, termed sirtuins, found in organisms ranging from bacteria to humans (Frye, R. A. (2000) Biochem. Biophys. Res. Commun. 273, 793-8). Sirtuins are highly conserved and contain a conserved catalytic domain of approximately 275 amino acids (Grozinger, C. M. et al., (2001) J. Biol. Chem. 276, 38837-38843). In S. cerevisiae alone, four additional homologues have been identified, while in humans, eight homologues have been identified (Grozinger, C. M. et al. (2001) supra). The yeast SIR2 gene shares the greatest similarity with genes found in other eukaryotes, where it is believed that these closely related homologues serve a comparable role in silencing. Interestingly, SIR2 and its homologues have been implicated in the genetic regulation of aging, both in yeast and C. elegans (Tissenbaum, H. A. & Guarente, L. (2001) Nature 410, 227-30; Sinclair, D. A. & Guarente, L. (1997) Cell 91, 1033-42) and in metazoan development though the details of how it affects these fundamental processes are still mysterious.
Recently, several groups (Luo, J. et al. (2001) Cell 107, 137-48; and Vaziri, H. et al. (2001) Cell 107, 149-59) have explored the influence of the mammalian homologues, Sir2α (the mouse homologue of S. cerevisiae SIR2, also known as mSIRT1) and SIR2α (the human homologue of S. cerevisiae SIR2, also known as hSIRT1), on the activity of the p53 tumor suppressor gene. These studies indicate that deacetylase activity of Sir2α and SIR2α act on p53, resulting in suppression of the tumor suppressor activity. They have also shown that this deacetylase activity is dependent on nicotinamide adenosine dinucleotide (NAD).
What is needed in the art, is a method for inhibiting the NAD+-dependent deacetylase activity of a member of the SIR2 family of proteins using a small molecule. Surprisingly, the present invention meets this need.